Geldanamycin analog 17-DMAG limits apoptosis in human peripheral blood cells by inhibition of p53 activation and its interaction with heat-shock protein 90 kDa after exposure to ionizing radiation

Abstract

Exposure to ionizing radiation induces p53, and its inhibition improves mouse survival. We tested the effect of 17-dimethylamino-ethylamino-17-demethoxygeldanamycin (17-DMAG) on p53 expression and function after radiation exposure. 17-DMAG, a heat-shock protein 90 (Hsp90) inhibitor, protects human T cells from ionizing radiation-induced apoptosis by inhibiting inducible nitric oxide synthase (iNOS) and subsequent caspase-3 activation. Using ex vivo human peripheral blood mononuclear cells, we found that ionizing radiation increased p53 accumulation, acute p53 phosphorylation, Bax expression and caspase-3/7 activation in a radiation dose- and time postirradiation-dependent manner. 17-DMAG inhibited these increases in a concentration-dependent manner (IC(50) = 0.93 ± 0.01 µM). Using in vitro models, we determined that inhibition of p53 by genetic knockout resulted in lower levels of caspase-3/7 activity 1 day after irradiation and enhanced survival at 10 days. Analysis of p53-Hsp90 interaction in ex vivo cell lysates indicated that the binding between the two molecules occurred after irradiation but 17-DMAG prevented the binding. Taken together, these results suggest the presence of p53 phosphorylation and Hsp90-dependent p53 stabilization after acute irradiation. Hsp90 inhibitors such as 17-DMAG may prove useful with radiation-based cancer therapy as well as for general radioprotection.

FIG. 1

Inhibition of radiation-induced p53 pathway by 17-DMAG. Jurkat cells were counted and seeded in fresh medium containing 10 µM 17-DMAG or vehicle. Cells were then irradiated 24 h after drug treatment and returned to the incubator. Cells were harvested 4 and 24 h postirradiation. Cell lysates were prepared and immunoblotting was performed to measure p53, Bax, Bcl-2 and actin levels. Intensities of 24-h bands were quantified by optical densitometry. Panel A: Representative immunoblots. Panel B: Quantification of p53 levels relative to actin. Panel C: Quantification of Bax levels relative to actin. Panel D: Quantification of Bcl-2 levels relative to actin. ns, nonsignificant.

FIG. 2

Inhibition of radiation-induced p53 expression by 17-DMAG is concentration-dependent in normal human PBMCs. Freshly isolated PBMCs from healthy human donor were counted and seeded in medium containing 17-DMAG at different concentrations for 24 h. Cells were then irradiated, returned to the incubator, and harvested 1, 4 and 24 h postirradiation. Total cell lysates from each sample were prepared for immunoblot analysis to determine the levels of p53 and actin. Protein levels were measured by optical densitometry. Panel A: Representative immunoblots. Panel B: Quantification of p53 levels relative to actin. *P < 0.05 and ***P < 0.001 compared with corresponding samples with no drug treatment (0 µM).

FIG. 3

17-DMAGinhibits radiation-induced increases in p53/Bax in normal human PBMCs. Freshly isolated PBMCs from healthy human subject were counted and seeded in medium containing 10 µM 17-DMAG or vehicle for 24 h. Cells were then irradiated, returned to the incubator, and harvested 1 h (for γ-H2AX and pp53 measurements), 4 h (for mRNA measurements), and 24 h (for p53/Bax/p21 protein) postirradiation. Panel A: Representative immunoblots showing effect of 17-DMAGon p53, Bax and p21 protein after irradiation. Total cell lysates from samples collected at 24 h were prepared for immunoblots to test p53, Bax, p21 and actin expression. Levels were measured using optical densitometry. Panel B: Quantification of p53 levels relative to actin. Panel C: Quantification of Bax levels relative to actin. Panel D: Quantification of p21 levels relative to actin. Panel E: Analysis of γ-H2AX levels 1 h postirradiation by flow cytometry. Panel F: Analysis of p53 phosphorylation levels on the serine residue at position 15; 1 h postirradiation by flow cytometry. Panel G: p53 gene expression. Total RNA was extracted from samples collected 4 h postirradiation and used for qRT-PCR analysis. Samples were tested for p53 expression levels using primers and probes specific to the human TP53 gene. Panel H: Bax gene expression. Total RNA used to test p53 gene expression was also studied by qRT-PCR using primers and probes specific to the human Bax gene. Results are presented as averages of triplicate samples from independent wells with standard errors. ns, nonsignificant.

FIG. 4

17-DMAG has no effect on Hsp90 levels but induces Hsp70 in concentration-dependent manner in normal human PBMCs. Freshly isolated PBMCs from a healthy human subject were counted and seeded for 24 h in medium containing 17-DMAG or vehicle. Cells were then irradiated, returned to the incubator, and harvested 24 h postirradiation. Total cell lysates from samples were prepared for immunoblots to test Hsp90, Hsp70 and actin levels. Levels were quantified by optical densitometry. Panel A: Representative immunoblots of Hsp90, Hsp70 and actin after pretreatment with different concentrations of 17-DMAG prior to irradiation with 8 Gy. Panel B: Quantification of Hsp90 and Hsp70 levels relative to actin shown in panel A. Panel C: Representative immunoblots of Hsp90, Hsp70 and actin in the presence or absence of 10 µM 17-DMAG prior to irradiation with 4 and 8 Gy. Panel D: Quantification of Hsp90 levels relative to actin shown in panel C. Panel E: Quantification of Hsp70 levels relative to actin shown in panel C. *P 0.05 and ***P 0.001 compared with corresponding samples with no drug treatment (0 µM). ns, nonsignificant.

FIG. 5

17-DMAG inhibits radiation-induced interaction between p53 and Hsp90 in normal human PBMCs. Freshly isolated PBMCs from healthy human subject were counted and seeded for 24 h in medium containing 10 µM 17-DMAG or vehicle. Cells were then irradiated with 8 Gy or left unirradiated, returned to the incubator, and harvested 4 h postirradiation. Total cell lysates were prepared and immunoprecipitated with anti-p53 antibody or control mouse IgG followed by immunoblot analysis with indicated antibodies. Total cell lysates were also tested by immunoblot analysis for protein content by probing with anti-actin antibody.

FIG. 6

Effect of 17-DMAG treatments on radiation-induced caspase-3/7 activity in normal human PBMCs. Freshly isolated PBMCs from healthy human subject were counted and seeded for 24 h in medium containing 10 µM 17-DMAG or vehicle. Cells were then irradiated with 4 or 8 Gy and returned to the incubator. Live cells were studied 24 h postirradiation for internal caspase-3/7 activities by Magic Red^® staining. Panel A: Representative images. Panel B: Quantification of fluorescence intensity.

FIG. 7

Direct correlation between p53 and apoptosis. Panel A: Representative immunoblot of p53 in TK6 and NH32 cells. NH32 is p53 knockout cell line derived fromTK6. Total cell lysates from both untreated cell lines were tested by immunoblot analysis with antibodies against p53 and actin. Panel B: Enhanced survival of NH32 after γ irradiation. Equal numbers of TK6 and NH32 cells were irradiated at the indicated doses or left unirradiated, and 5,000 cells were assessed by clonogenic assay. Numbers of colonies were counted on day 10, and data are shown as fractions of the corresponding nonirradiated controls. Results are averages of triplicate data with standard deviations. Panel C: Representative images of caspase-3/7 activity in TK6 and NH32 cells. Live cells were studied 24 h postirradiation for internal caspase-3/7 activities by Magic Red® staining. Panel D: Quantification of fluorescence intensity. *P < 0.05 and ***P < 0.001 compared between different cell lines at each radiation dose.